1) The role of pyrin in resistance to Yersinia pestis Mutations in MEFV, encoding the pyrin protein, cause familial Mediterranean fever (FMF). The carrier frequency of FMF-associated MEFV mutations is extraordinarily high in Mediterranean and Middle Eastern populations, suggesting that heterozygous FMF mutations may confer a selective advantage against some pathogenic microbes. Inactivation of the RhoA GTPase is a common bacterial virulence mechanism, because RhoA activation is required for actin polymerization and leukocyte migration, phagocytosis, and degranulation. The pyrin inflammasome senses RhoA inactivation and activates IL-1beta as part of the host defense against these bacteria. Pathogenic Yersinia, including Y. pestis, the agent of plague in humans, deliver virulence effectors, termed Yersinia outer proteins (Yops), into host cells; some of these Yops are known to inactivate RhoA. In the current reporting period, we collaborated with Dr. James Bliska at Stony Brook University to test the hypothesis that FMF-associated pyrin mutations might confer a selective advantage against Yersia species. We utilized pyrin knockin (KI) and knockout (KO) mice to examine the role of pyrin in host defense against Yersinia infection. In Yersinia-infected macrophages, the pyrin inflammasome was activated by the RhoA-inactivating enzymatic activities of YopE and YopT. YopM specifically inhibited pyrin to promote virulence by activating the host protein kinases (PKN1 and PKN2) that phosphorylate pyrin to block pyrin inflammasome activation. We found that Pyrin-KO mice were highly susceptible to Yersinia YopM deletion mutant infection, while WT mice were not susceptible. However, bone marrow-derived macrophages (BMDMs) from both homozygous and heterozygous FMF-KI mice released significantly higher levels of IL-1beta in comparison with WT BMDMs in response to Yersinia infection. These results suggest that the FMF-associated mutant pyrin is not suppressed by YopM, thereby providing survival advantage of FMF-KI mice against Yersinia infection. We further found that FMF-KI mice, both homozygotes and heterozygotes, showed significant resistance to Y. pestis infection in comparison with WT mice. These findings, taken together with the historical record of high-mortality Yersinia epidemics, suggest that Yersinia pestis played an important role in selecting for the high frequency of FMF-associated MEFV mutations in Mediterranean and Middle Eastern populations. We are currently preparing a manuscript describing these findings. 2) Characterization of a recessively inherited autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme We studied 9 patients with bi-allelic mutations in TRNT1 and the syndrome of congenital sideroblastic anemia with immunodeficiency, fever, and developmental delay (SIFD). We identified 8 mutations in these 9 patients, 3 of which had not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly IL-6, interferon-gamma, and interferon-induced cytokines were elevated in the serum, whereas TNF and IL-1beta were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients fibroblasts showed significant deficiency of mature cytosolic tRNAs. Electron microscopy of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for transfusions, and improved growth. The findings of this study indicate that mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease manifestations. A manuscript describing these findings has been submitted during the current reporting period. 3) Examination of the Effectiveness of Tumor Necrosis Factor (TNF) Inhibitors in the Deficiency of Adenosine Deaminase 2 (DADA2) DADA2 is a recessively inherited disorder characterized by recurrent fevers, livedo racemosa, early-onset lacunar strokes, and, in some patients, polyarteritis nodoasa (PAN). It is caused by mutations in CECR1, encoding ADA2. In a retrospective analysis of 15 patients referred to our clinic in whom we made the diagnosis of DADA2 and who had been treated with a wide spectrum of potent immunosuppressives (but not TNF inhibitors), patients had an average of 1 stroke every 21 months. Based on our previous demonstration of perivascular TNF in DADA2 biopsy specimens and the favorable response of Israeli DADA2 patients with PAN to TNF inhibitors, we compared the frequency of stroke in 15 NIH DADA2 patients before and after initiation of TNF inhibitors (etanercept, infliximab, adalimumab). Before anti-TNF initiation, these 15 patients had 55 strokes over 2077 patient months (model based recurrence rate 0.025) compared to 0 strokes over 583 patient months after initiation of anti-TNF (recurrence rate 0; P = 6.7 x 10e-8). A matched follow up time analysis using 583 patient months both prospectively and retrospectively estimates the probability of stroke before initiation of anti-TNF as opposed to afterward to be 1 (Blyth-Still-Casella exact confidence interval = 0.9147, 1; P = 2.9 x 10e-11). We also examined the frequency of hemorrhagic strokes in DADA2 patients prior to their referral to the NIH, stratified by whether they had received aspirin and/or anticoagulants. The proportions of patients with hemorrhagic strokes on no antiplatelet or anticoagulant treatment, aspirin alone, or aspirin plus other anticoagulants and/or antiplatelet agents, were 0/9, 0/6, and 4/7, respectively (P = 0.007, Fischers exact test). Our data strongly support the use of TNF inhibitors with a history of stroke, but argue against the use of aspirin plus other anticoagulants in these patients. We are currently preparing a manuscript describing these findings. 4) Characterization of a family with early-onset systemic lupus erythematosus (SLE) Using whole-exome sequencing and single nucleotide polymorphism array genotyping in a multiplex consanguineous Turkish family with SLE, we identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using Sanger sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old-girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enhancement of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA-haplotypes were not shared among the 4 affected subjects. This study demonstrates a novel mutation in C1R as the cause of monogenic SLE, with disease severity influenced by additional common and rare genetic variants. A manuscript describing these findings was published in Arthritis and Rheumatology during the reporting period.